Biopolymers, such as polypeptides and polynucleotides, are routinely synthesized by solid-phase methods in which polymer subunits are added stepwise to a growing polymer chain immobilized on a solid support. For polynucleotides and polypeptides, this general synthetic procedure can be carried out with commercially available synthesizers that construct the biopolymers with defined sequences in an automated or semi-automated fashion. However, commercially available synthesizers do not allow the efficient synthesis of oligosaccharides; typically, the yields of oligosaccharides synthesized using the commercially available apparatus are poor.
The glycosylation reaction is one of the most thoroughly studied transformations in organic chemistry. In the most general sense, a glycosylation is the formation of an acetal connecting two sugar units. The majority of glycosylating agents follow similar paths of reactivity. The anomeric substituent acts as a leaving group thereby generating an electrophilic intermediate. Reaction of this species with a nucleophile, typically a hydroxyl group, leads to the formation of a glycosidic linkage. This reaction may proceed via a number of intermediates depending on the nature of the leaving group, the activating reagent and the solvent employed.
Glycosyl trichloroacetimidates, thioglycosides, glycosyl sulfoxides, glycosyl halides, glycosyl phosphites, n-pentenyl glycosides and 1,2-anhydrosugars are among the most reliable glycosyl donors (See FIG. 1). Despite the wealth of glycosylating agents available, no single method has been distinguished as a universal donor. Contrary to peptide and oligonucleotide synthesis, the inherent differences in monosaccharide structures make it unlikely that a common donor will prevail. Rather, individual donors will see use in the construction of certain classes of glycosidic linkages.
Solid-Phase Chemical Synthesis
Solution-phase oligosaccharide synthesis remains a slow process due to the need for iterative coupling and deprotection steps with purification at each step along the way. To alleviate the need for repetitive purification events, solid-phase techniques have been developed. In solid-phase oligosaccharide synthesis there are two methods available (See FIGS. 2 and 3). The donor-bound method links the first sugar to the polymer through the non-reducing end of the monomer unit (FIG. 2). The polymer-bound sugar is then converted into a glycosyl donor and treated with an excess of acceptor and activator. Productive couplings lead to polymer bound disaccharide formation while decomposition products remain bound to the resin. Elongation of the oligosaccharide chain is accomplished by converting the newly added sugar unit into a glycosyl donor and reiteration of the above cycle. Since most donor species are highly reactive, there is a greater chance of forming polymer-bound side-products using the donor-bound method.
Alternatively, acceptor bound strategies have found considerable use in solid-phase oligosaccharide synthesis (FIG. 3). In this approach, the first sugar is attached to the polymer at the reducing end. Removal of a unique protecting group on the sugar affords a polymer-bound acceptor. The reactive glycosylating agent is delivered in solution and productive coupling leads to polymer-bound oligosaccharides while unwanted side-products caused by donor decomposition are washed away. Removal of a unique protecting group on the polymer-bound oligosaccharide reveals another hydroxyl group for elongation.
While the merits of the donor-bound method have been demonstrated by Danishefsky and co-workers, the most popular and generally applicable method of synthesizing oligosaccharides on a polymer support remains the acceptor-bound strategy. For a review, see: P. H. Seeberger, S. J. Danishefsky, Acc. Chem. Res., 31 (1998), 685. The ability to use excess glycosylating agents in solution to drive reactions to completion has led to widespread use of this method. All of the above mentioned glycosylating agents have been utilized with the acceptor-bound method to varying degrees of success.
Increasingly, there is an interest in the automated synthesis of oligosaccharides. For example, it is often of interest, in examining structure-function relationships involving sugars, to generate a mixture of oligosaccharides having different residues at a particular position or varying in anomeric configuration at a glycosidic linkage. As another example, oligosaccharides having a desired activity, such as a high binding affinity to a given receptor or antibody, may be identified by (a) generating a large number of random-sequence oligosaccharides, and (b) screening these oligosaccharides to identify one or more oligosaccharides having the desired binding affinity.
Current apparatus for synthesizing oligosaccharides are limited in both the number and quantity of the oligosaccharides which can be synthesized. These limitations have restricted the availability of oligosaccharides, both for structure-function studies, and for selection methods.